Endonuclease heteroduplex mismatch cleavage for detecting mutation genetic variation of trypsin inhibitors in soybean

102 G. Petrović et al.
Pesq. agropec. bras., Brasília, v.49, n.2, p.102-108, fev. 2014
DOI: 10.1590/S0100-204X2014000200004
Endonuclease heteroduplex mismatch cleavage for detecting
mutation genetic variation of trypsin inhibitors in soybean
Gordana Petrović(1)
, Zorica Nikolić(1)
, Vuk Đorđević(1)
, Vesna Župunski(1)
,
Dušica Jovičić(1)
, Maja Ignjatov(1)
and Dragana Milošević(1)
(1)
Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, Serbia. E‑mail: gordana.zdjelar@nsseme.com,
zorica.nikolic@nsseme.com, vuk.djordjevic@nsseme.com, dusicajovicic@hotmail.com, maja.ignjatov@nsseme.com, dragana.petrovic@nsseme.com
Abstract – The objective of this work was to evaluate the genetic variation of trypsin inhibitor in cultivated
(Glycine max L.) and wild (Glycine soja Siebold & Zucc.) soybean varieties. Genetic variations of the Kunitz
trypsin inhibitor, represented by a 21‑kD protein (KTI), and of the Bowman‑Birk trypsin–chymotrypsin inhibitor
(BBI) were evaluated in cultivated (G. max) and wild (G. soja) soybean varieties. Endonuclease heteroduplex
mismatch cleavage assays were performed to detect mutations in the KTI gene, with a single‑stranded specific
nuclease obtained from celery extracts (CEL I). The investigated soybean varieties showed low level of genetic
variation in KTI and BBI. PCR‑RFLP analysis divided the BBI‑A type into subtypes A1 and A2, and showed
that Tib type of KTI is the dominant type. Digestion with restriction enzymes was not able to detect differences
between ti‑null and other types of Ti alleles, while the endonuclease heteroduplex mismatch cleavage assay
with CEL I could detect ti‑null type. The digestion method with CEL I provides a simple and useful genetic tool
for SNP analysis. The presented method can be used as a tool for fast and useful screening of desired genotypes
in future breeding programs of soybean.
Index terms: Glycine max, Glycine soja, antinutritional factors, protease inhibitors, SNP.
Endonuclease com incompatibilidade heteroduplex para detectar
mutação e variações genéticas de inibidores da tripsina em soja
Resumo – O objetivo deste trabalho foi avaliar a variação genética do inibidor de tripsina em variedades
cultivadas (Glycine max) e silvestres (Glycine soja) de soja. Foram avaliadas as variações genéticas do
inibidor de tripsina Kunitz, representado pela proteína 21‑kDa (KTI), e do inibidor de tripsina‑quimotripsina
Bowman‑Birk (BBI), em variedades de soja cultivadas (G. max) e selvagens (G. soja). Ensaios de clivagem
foram feitos com endonuclease de incompatibilidade heteroduplex, para a detectar mutações no gene de
KTI, com uma única nuclease específica de cadeia simples, obtida a partir de extractos de aipo (CEL I).
As variedades de soja estudadas apresentaram baixo nível de variação genética em KTI e BBI. A análise por
PCR ‑RFLP dividiu o BBI‑A em A1 e A2 e mostrou que o Tib do KTI é o tipo dominante. A digestão com
enzimas de restrição não foi capaz de detectar diferenças entre os tipos de ti‑null e outros alelos Ti, enquanto
o ensaio com endonucleases com incompatibilidade heteroduplex com CEL I pôde detectar o tipo ti‑null.
O método de digestão com CEL I fornece uma ferramenta genética simples e útil para a análise de SNP.
O método apresentado pode ser utilizado como ferramenta para a triagem rápida e útil de genótipos desejáveis
em futuros programas de melhoramento de soja.
Termos para indexação: Glycine max, Glycine soja, factores antinutritionais, inibidores de protease, SNP.
Introduction
In general, most commercial soybean cultivars
contain about 40% protein and represent an important
source of this nutrient (Song et al., 2013). Soybean
proteins have traditionally been used for animal
feed; however, their use for human consumption are
increasing (Livingstone et al., 2007). Nevertheless,
raw soybean cannot be used for animal feeding
because of the presence of some antinutritional factors
that decrease its nutritional value. Protease inhibitors,
which represent about 6% of the total seed protein
content, are among the main antinutritional factors
in soybean seeds (Wang et al., 2004). There are two
major classes of protease inhibitors: the Kunitz trypsin
inhibitor (KTI), represented by a 21‑kD protein, and
the Bowman‑Birk trypsin–chymotrypsin inhibitor
(BBI), which consists of several related 8‑kD proteins

Endonuclease heteroduplex mismatch cleavage for detecting 103
Pesq. agropec. bras., Brasília, 49, n.2, p.102-108, fev. 2014
DOI: 10.1590/S0100-204X2014000200004
(Kim et al., 2010). Approximately 80% of the trypsin
inhibition activity is caused by KTI (Barros et al.,
2008).
The biological roles of protease inhibitors are not
clear. A number of functions has been proposed for
BBIs, including the regulation of protease activity
during seed germination and the protection of plants
from insects and microorganisms. Moreover, BBIs may
also function as storage of sulfur amino acids (Barros
et al., 2012; Cruz et al., 2013). Recent investigations
have focused on its medicinal utility for suppressing
both initiation and promotion stages of carcinogenesis
(Rakashanda & Amin, 2013). BBI protease inhibitors
are double‑headed serine protease inhibitors that bind
both enzymes at two independent reactive sites with a
network of highly conserved disulfide bridges (Barros
et al., 2012).
Genes encoding BBI in Glycine max and Glycine
soja are a multigene family, with at least ﬁve members:
BBI‑A, BBI‑B, BBI‑CII, BBI‑DII and BBI‑EI. BBI‑B
is supposed to be encoded by a gene which is closely
related to BBI‑A, designated as BBI‑A2, and both are
very similar. Post‑translational proteolysis indicates
that BBI‑EI is originated from BBI‑DII. Thus, BBIs
are grouped in three types with distinct characteristics:
BBI‑A, BBI‑C, and BBI‑D (Deshimaru et al., 2004;
Wang et al., 2008; Barros et al., 2012).
Based on electrophoretograms, 12 forms of KTI
have been found: Tia and Tib (Wang et al., 2008, 2010),
Tic (Hymowitz, 1973), Tid (Zhao & Wang, 1992), Tie
(Wang et al., 2008), Tif (Wang et al., 2004), Tibi5
(Wang
et al., 2008), Tiaa1
, Tiaa2
, Tiab1
, Tig (Wang et al., 2008)
and ti‑null type (Orf & Hymowitz,1979). Of these,
Tia and Tib, which differ by nine amino acids, are the
predominant types (Lee et al., 2012). These several
polymorphic soybean KTI types are controlled by
codominant multiple alleles at a single locus (Wang
et al., 2008). There are at least ten distinct DNA
sequences in soybean genome for the Kunitz trypsin
inhibitor, some of which occur in tandem pairs (Jofuku
& Goldberg, 1989). At least three of these have been
conﬁrmed to represent functional genes, referred to
as KTI1, KTI2 and KTI3. The major Kunitz trypsin
inhibitor gene in soybean seeds is KTI3 (Jofuku &
Goldberg 1989). The null phenotype of KTI, which has
reduced amounts of Kunitz trypsin inhibitor and lacks
detectable Kunitz trypsin inhibitor activity, is inherited
as a recessive allele ti (Orf & Hymowitz, 1979). This
null line has three mutations: two deletions and one
G → T transversion occurred within the KTI3 gene
(Jofuku et al., 1989). These mutations cause a
translational frameshift that results in four stop codons
to be inserted into the KTI3 mRNA reading frame and
premature termination of KTI3 mRNA translation,
and leads to a 100‑fold reduction of KTI3 mRNA in
soybean embryos (Jofuku et al., 1989).
There are some correlations between a single
nucleotide polymorphism (SNP) and many of the
key characters of crops. Even the phenotype of some
characters can be indicated by SNPs. Therefore,
detecting SNPs for important functional genes and
identifying their relationship with desired phenotypes
are important tools in plant breeding program.
According to the demand, it may be necessary to
develop soybean cultivars with a high content of
inhibitors for resistance to insects, or to develop
cultivars with a reduced content of inhibitors for a
better nutritive value. One of the methods for SNP
detection is a heteroduplex mismatch cleavage assay
using the endonuclease CEL I from celery in plants
(Zolala et al., 2009), animals (Kuroyanagi et al.,
2013), and humans (Till et al., 2006). CEL I is a
mannosyl glycoprotein which cuts the 3’ side of the
loops formed in double‑stranded heteroduplex DNA
molecules, at sites of base substitutions, and small
insertion or deletions (indels) (Yang et al., 2004). CEL
I endonuclease assay has proved to be useful for SNP
detection in tomato (Yang et al., 2004), wheat (Chen
et al., 2011), common bean (Galeano et al., 2009), and
sunflower (Fusari et al., 2011).
The objective of this work was to evaluate the genetic
variation of trypsin inhibitor in cultivated (Glycine
max), and wild (Glycine soja) soybean varieties.
Materials and Methods
Ten soybean varieties were selected, five of them
from USDA (Altona, Wilkin, Amsoy 71, Panther,
and Kunitz); two from the Institute of Field and
Vegetable Crops, Soybean Breeding Program from
Novi Sad, Serbia ('Vojvodjanka' and 'Fortuna'); and
three were wild soybean varieties (G. Soja) provided
by N.I. Vavilov Research Institute collection, from
St. Petersburg, Russia (37‑2, Primorye district, Russia;
42‑2, Khabarovsk district, Russia; and 7‑18, Amur
region, Russia. A DNeasy plant mini kit (Qiagen,
Germany) for genomic DNA extraction, according
to the manufacturer’s manual, was used. Quality and

DOI: 10.1590/S0100-204X2014000200004
quantity of the extracted DNA was checked with a
UV/VIS spectrophotometer (Genesys 10S, Thermo
Scientific, USA). The A260/A280 of extracted DNA
ranged from 1.7‑2.0.
Isoinhibitor‑specific oligonucleotide primers used
for PCR amplification of BBI‑A, BBI‑C, and BBI‑D
are reported in Table 1, as well as the oligonucleotide
primers for KTI3 gene. Endogenous gene lectin was
used as quality control for DNA and PCR efficiency.
PCR was carried out using premix of 2x PCR
Master Mix (Fermentas, Vilnius, Lithuania), with final
concentration of 2 mmol L‑1
MgCl2, 0.2 mmol L‑1
dNTP,
and 1.25 units Taq DNA Polymerase (recombinant).
PCR was performed in a final volume of 25 µL with
0.2 pmol µL‑1
primers and approxmately 50 ng DNA.
Amplifications were carried out in a Mastercycler
ep gradient S thermal cycler (Eppendorf, Hamburg,
Germany) under the following touchdown program:
initial denaturation at 94°C for 3 min, followed by
a touchdown program for 7 cycles, with successive
annealing temperature decrements of 1°C in every
cycle.Forthesefirst7 cycles,thereactionwasdenatured
at 94°C for 50 s, followed by annealing at 62°C →
56°C for 50 s, and polymerization at 72°C for 1 min
and 30 s. The 30 subsequent cycles for amplification
were similar, except for annealing temperature, which
was 56°C for 50 s.
Amplified fragments for BBI‑A were further
subjected to digestion by HindIII restriction enzyme
(Fermentas, Vilnius, Lithuania) (Deshimaru et al.,
2004), while amplified fragments for KTI3 were
digested with Mse I (Tru1 I) restriction enzyme
(Fermentas, Vilnius, Lithuania) (Wang et al., 2008).
Amplification and restriction fragments were
determined using electrophoresis on 2% agarose
gel containing ethidium bromide (0.5 g mL‑1
). The
expected size of the amplified fragments was estimated
by comparison with O’RangeRuler 50 bp DNA Ladder
and FastRuler DNA Ladder, Low Range (Fermentas,
Vilnius, Lithuania).
The agarose gel was visualized using UV
transilluminator, and the images were captured with
DOC II PRINT system (Vilber Lourmat, Marne-la-
Valleé, France).
Plant juice extracts with CELI activity were prepared
as described byTill et al. (2006), for purification of CEL
I. Only the extraction, salting out, and dialysis steps of
the purification protocol were performed. Store‑bought
celery stalks (about 0.7 kg) were juiced at 4°C. Celery
juice was adjusted to reach a final concentration of
0.1 mol L‑1
Tris‑HCl, pH 7.7, 100 µmol L‑1
PMSF
and 0.01% Triton X‑100. The obtained solution was
then centrifuged for 20 min at 10,000 g to pellet
debris. Supernatant was brought to 25% saturation in
(NH4)2SO4, mixed for 1 hour at 4°C, and centrifuged at
10,000 g, at 4°C for 45 min. Resulting supernatant was
adjusted to 80% with (NH4)2SO4, mixed for 1 hour at
4°C, and centrifuged at 10,000 g for 1.5 hour. Pellet was
suspended in buffer with 0.1 mol L‑1
Tris‑HCl pH 7.7,
100 µmol L‑1
PMSF, 0.01% Triton X‑100 (1/10 of
starting plant juice extract volume). Suspension was
dialyzed against the same buffer over night. Extract
aliquots were stored at aproximately 20°C.
To form the heteroduplex, KTI PCR products from
Kunitz variety – lacking active KTI – and from other
soybean varieties were mixed in 1:1 ratio and subjected
to heating and re‑annealing process, running the
following program: 95°C for 2 min; 95°C ramping to
85°C (‑2°C per second); 85°C ramping to 25°C (‑0.3°C
per second); and 4°C hold, to form heteroduplex.
For CEL I digestion, the 10 µL of heteroduplexes
were incubated in 5 µL of buffer D (20 mmol L‑1
Tris–
HCl,pH 7.4,25 mmolL‑1
KCl,10 mmolL‑1
MgCl2)with
5 µLpurified plant extract with CEL I (0.01 µg) at 45°C
for 35 min (Oleykowski et al., 1998). The reaction was
stopped with 5 μL of 0.15 mol L‑1
EDTA. The digested
products were determined using electrophoresis on 2%
agarose gel, as previously described.
Results and Discussion
Soybean KTI has several polymorphic types, which
are controlled by codominant multiple alleles at a
single locus (Wang et al., 2008). Three of the KTI
Table 1. Oligonucleotide primers used for PCR amplification
of lectin, KTI3, BBI‑A, BBI‑C, and BBI‑D genes.
Gene Sequence (5’→3’) Amplicon
(bp)
Reference
Lectin
GACGCTATTGTGACCTCCTC
GAAAGTGTCAAGCTTAACAGCGACG
318
Meyer et al.
(1996)
KTI3
AGTCCCGATTCTCCCAACA
AGTACTCTCACACTTGTGTC
700
Jofuku et al.
(1989)
BBI‑A
ACATGGTGGTGCTAAAGGTGTGTT
CTTGTTCATTAGTAGTTTTCCTTGTCA
350
Wang et al.
(2008)
BBI‑C
GACACTTGACAGGAAAAACAG
GCCAAAAGCAAATTACTGGCC
480
Wang et al.
(2008)
BBI‑D
ACAGCAAAAACAACTAATAAAG
TAAAAATGACCAAATTTGCT
550
Wang et al.
(2008)

DOI: 10.1590/S0100-204X2014000200004
genes (KTI1, KTI2, and KTI3) have been cloned and
sequenced (Krishnan, 2001). A sequence for KTI was
amplified by PCR (Figure 1) using a set of two primers
designedonthebasisofDNAsequencesofKTI3(= Tia)
(Jofuku et al., 1989). A fragment of aproximately 700
bp was amplified in all analyzed varieties from the
USDA germplasm collection, including the Kunitz
variety – a genotype lacking active trypsin inhibitor.
The null phenotype of KTI is due to a mutation
in the Kunitz trypsin inhibitor structural gene, and
it is inherited as a recessive allele ti. It has reduced
amounts of Kunitz trypsin inhibitor protein and lack
detectable Kunitz trypsin inhibitor activity (Orf &
Hymowitz, 1979). Therefore, one of the methods
for detecting mutation and SNP is a heteroduplex
mismatch cleavage assay using the endonuclease CEL
I from celery (Zolala et al., 2009). The ability of celery
juice extract CEL I to detect a mismatch at one or more
nucleotide positions, without prior knowledge about
this sequence, was shown by Oleykowski et al. (1998).
The laboratory has purified this enzyme according to
Till et al. (2006), which made the mutation detection
assay less expensive. The enzyme is found to be
extremely stable during purification, storage, and
assay. Upon digestion of formed heteroduplexes with
CEL I enzyme, the digested products were visualized
in 2% agarose gels, avoiding the need for labeled
primers, polyacrylamide gels, and DNA sequencers
used in earlier versions of the methods (Galeano et al.,
2009). The method for detecting SNPs in stress‑related
genes in rice, using CEL I and agarose gels, provide
results which perfectly corresponded to the ones
from polyacrylamide‑ and LI‑COR‑based analyses
(Raghavan et al., 2007). Chen et al. (2011) also showed
that agarose gels could be convenient for detecting
SNP in common wheat. Moreover, the PCR reaction
is cheaper because the primers used are not labelled.
Digestion of the heteroduplexes formed for KTI
gene with CEL I enzyme generated bands with 500 bp
and approximately 250 bp, in addition to full‑lenght
uncleaved product of 700 bp (Figure 2). The sum of
the cleaved fragments is theoretically about the length
of the PCR product (Oleykowski et al., 1998). Two
obtained fragments (Figure 2) indicate the presence of
SNP in Kunitz variety KTI gene, confirming that assay
with the CEL I crude extract isolated in laboratory has
the potential to identify SNP mismatches.
Restriction enzyme Mse I (Tru1 I) was used to
determine which KTI type was present. Tia type alleles
had two restriction sites resulting in three fragments.
Tib type had one restriction site resulting in two
fragments (Wang et al., 2008). Tib type was found in
cultivated and in wild soybean varieties (Figure 3).
Electrophoretic forms Tia, Tib, Tic, and Tid have been
reported in cultivated soybeans (Hymowitz, 1973;
Figure 1. Agarose gel electrophoresis of PCR products for
KTI. M1, O’RangeRuler, 50 bp DNA Ladder, 50‑1000 bp;
1. blank, no template control; 2. negative control, maize;
3. positive control, 'Vojvodjanka'; 4. 'Altona'; 5. 'Wilkin';
6. 'Amsoy 71'; 7. 'Panther'; 8. 'Kunitz'; 9. 'Vojvodjanka';
10. 'Fortuna'; 11. Glycine soja 37‑2; 12. Glycine soja
42‑2; 13. Glycine soja 7‑18; M2
, Low Range DNA Ladder,
50‑1500 bp.
Figure 2. Agarose gel electrophoresis of heteroduplex
mismatch cleavage CEL I endonuclease products in KTI
gene. Mutants could be identified as those products that
showed cleaved bands (500 bp and 250 bp), in addition to the
full‑length, uncleaved product (700 bp). M1, O’RangeRuler,
50 bp DNA Ladder, 50‑1000 bp; 1. 'Kunitz' vs. 'Altona';
2. 'Kunitz' vs. 'Wilkin'; 3. 'Kunitz' vs. 'Amsoy' 71; 4. 'Kunitz'
vs. 'Panther'; 5. 'Kunitz' vs. 'Vojvodjanka'; 6. 'Kunitz' vs.
'Fortuna'; 7. 'Kunitz' vs. Glycine soja 37‑2; 8. 'Kunitz' vs.
Glycine soja 42‑2; 9. 'Kunitz' vs. Glycine soja 7‑18; M2
,
Low Range DNA Ladder, 50‑1500 bp.

DOI: 10.1590/S0100-204X2014000200004
Zhao & Wang, 1992). Hymowitz (1973) determined
that 89% of USDA soybean collection contained
the Tia variant. In contrast, Tic was found only in
0.3% of the collection, and it commonly exists in
cultivated soybeans (Wang et al., 2008). Wang et al.
(2010) suggests that both wild and cultivated soybean
usually contain the most commonly occurring Tia and
Tib types, while the Tid form was found in just one
Chinese cultivar. Lee et al. (2012) also found Tia and
Tib as the predominant types. Unlike the digestion
with CEL I, amplification of KTI products, followed
by digestion with restriction enzymes, was not able to
detect differences between ti‑null and other types of Ti
alleles.
Based on their structural features and inhibitory
characteristics, BBIs are grouped in three main types –
BBI‑A, BBI‑C, and BBI‑D – and consist in a multigene
family (Deshimaru et al., 2004). Using cultivated
soybean and wild soybean genomic DNA as templates,
fragments of 350 bp for BBI‑A, 480 bp for BBI‑C, and
550 bp for BBI‑D were amplified (Figure 4). There
were no variations in the amplificon sizes among
cultivated and wild soybean samples, for all three
inhibitors. Based on sequence comparisons, Wang
et al. (2008) suggests that both wild and cultivated
soybean had similar BBI genes. This is probably due
to the close phylogenetic relation between the two
species (Deshimaru et al., 2004).
BBI‑A was further divided into two subtypes –
A1 and A2 – according to small differences in their
nucleotide sequences (Deshimaru et al., 2004).
Amplified fragments for BBI‑A were further subjected
to digestion by HindIII restriction enzyme (Figure 5),
for which only the coding sequence for BBI‑A1
contains a clevage site, resulting in two fragments
(Deshimaru et al., 2004). Between the USA‑origin
soybean, only 'Amsoy 71' showed the presence of
Figure 3. PCR‑RFLP profiles by Mse I (Tru1 I) restriction
digest of the KTI gene. Tib type has one restriction site. M1,
O’RangeRuler, 50 bp DNA Ladder, 50‑1000 bp; 1. 'Altona';
2. 'Wilkin'; 3. 'Amsoy' 71; 4. 'Panther'; 5. 'Kunitz'; 6.
'Vojvodjanka'; 7. 'Fortuna'; 8. Glycine soja 37‑2; 9. Glycine
soja 42‑2; 10. Glycine soja 7‑18; M2
, Low Range DNA
Ladder, 50‑1500 bp.
Figure 4. Agarose gel electrophoresis of PCR products for
BBI‑A. M1, O’RangeRuler, 50 bp DNA Ladder, 50‑1000
bp; 1. blank, no template control; 2. negative control,
maize; 3. 'Altona'; 4. 'Wilkin'; 5. 'Amsoy 71'; 6. 'Panther';
7. 'Kunitz'; 8. 'Vojvodjanka'; 9. 'Fortuna'; 10. Glycine soja
37‑2; 11. Glycine soja 42‑2; 12. Glycine soja 7‑18; M2
, Low
Range DNA Ladder, 50‑1500 bp.
Figure 5. PCR‑RFLP profiles by HindIII restriction digest
of the BBI‑A gene. BBI‑A1 subtype contains a clevage
site. M1, O’RangeRuler, 50 bp DNA Ladder, 50‑1000
bp; 1. 'Altona'; 2. 'Wilkin'; 3. 'Amsoy 71'; 4. 'Panther';
5. 'Kunitz'; 6. 'Vojvodjanka'; 9. 'Fortuna'; 10. Glycine soja
37‑2; 11. Glycine soja 42‑2; 12. Glycine soja 7‑18; M2
, Low
Range DNA Ladder, 50‑1500 bp.

DOI: 10.1590/S0100-204X2014000200004
A2 subtype, while two varieties from the Institute of
Field and Vegetable Crops had this subtype. Out of the
three wild soybean genotypes, just one had A1 subtype
(Figure 5). Deshimaru et al. (2004) suggests that these
two subtypes for BBI‑A occur from distinct genes in
the wild soybean genome, and not from polymorphic
alleles in the genome.
Conclusions
1. There is a low level of genetic variation in 21‑kD
protein (KTI) and Bowman‑Birk trypsin–chymotrypsin
inhibitor (BBI) between the investigated varieties of
cultivated and wild soybean.
2. The digestion of KTI products with restriction
enzymes show that Tib type of KTI is a dominant type
among the analyzed varieties, but it is not able to detect
differences between ti‑null and other types of Ti alleles.
3. The digestion method with celery extracts (CEL I)
described here provide a simple and useful genetic tool
for single nucleotide polymorphism (SNP) analysis.
Acknowledgments
To the Ministry of Education, Science and
Technological Development of the Republic of Serbia,
for support, by the projects TR‑31024 and TR‑31022.
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Received on October 2, 2013 and accepted on January 30, 2014

Endonuclease heteroduplex mismatch cleavage for detecting mutation genetic variation of trypsin inhibitors in soybean

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